Michael A. Lawton

Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to -173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyltransferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding. These data suggest that SBF-1 is identical to, or closely related to, GT-1.

Cloning and properties of a rice gene encoding phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL) genomic sequences were isolated from a rice (Oryza sativa L.) genomic library using a PCR-amplified rice PAL DNA fragment as a probe. There is a small family of PAL genes in the rice genome. The nucleotide sequence of one PAL gene, ZB8, was determined. The ZB8 gene is 4660 bp in length and consists of two exons and one intron. It encodes a polypeptide of 710 amino acids. The transcription start site was 137 bp upstream from the translation initiation site. Rice PAL transcripts accumulated to a high level in stems, with lower levels in roots and leaves. Wounding of leaf tissues induced ZB8 PAL transcripts to a high level. In rice suspension-cultured cells treated with fungal cell wall elicitors, the ZB8 PAL transcript increased within 30 min and reached maximum levels in 1–2 h. The transcription of the ZB8 gene was investigated by fusing its promoter to the reporter gene β-glucuronidase (GUS) and transforming the construct into rice and tobacco plants, as well as rice suspension-cultured cells. High levels of GUS activity were observed in stems, moderate levels in roots and low levels in leaves of transgenic rice and tobacco plants. Histochemical analysis indicated that in transgenic rice the promoter was active in root apical tips, lateral root intiation sites, and vascular and epidermal tissues of stems and roots. In rice flowers, high GUS activity was observed in floral shoots, receptacles, anthers and filaments, occasionally GUS activity was also detected in lemma and awn tissues. In tobacco flowers, high GUS activity was detected in the pink part of petals. Consistent with the activity of endogenous PAL transcripts, wounding of rice and tobacco leaf tissues induced GUS activity from low basal levels. Tobacco mosaic virus (TMV) infection of tobacco leaves induced GUS activity to a high level. Fungal cell wall elicitors strongly induced GUS activity and GUS transcripts to high levels in transgenic rice suspension-cultured cells. We demonstrated that the promoter of ZB8 gene is both developmentally regulated and stress-inducible.

Expression of a soybean β-conclycinin gene under the control of the Cauliflower Mosaic Virus 35S and 19S promoters in transformed petunia tissues.

A gene encoding the α′-subunit of β-conglycinin was ligated to the 19S and 35S promoters of Cauliflower Mosaic Virus and introduced into petunia plants on a disarmed Ti-plasmid using Agrobacterium tumefaciens. Transformed cells were regenerated into whole plants and ummunoreactive polypeptides and hybridizable, polyadenylated mRNA were detected in transformed tissues. Expression from the 35S promoter was 10 to 50 times greater than expression from the 19S promoter. The level of immunodetectable polypeptides was greater in seeds than in leaves or callus tissue. In addition, the pattern of α′-polypeptide breakdown products was distinctive in seeds and leaves. We conclude that in seeds the higher levels of the α′-polypeptide reflect enhanced stability of this protein.

Glutathione-elicited changes in chromatin structure within the promoter of the defense gene chalcone synthase

Sites hypersensitive to digestion by DNase I have been identified within the 5′-flanking and 3′-coding sequences of genes encoding the defense enzyme chalcone synthase in bean (Phaseolus vulgaris L.). Two of the 5′-flanking hypersensitive sites are markedly induced upon elicitation of cells with glutathione and delineate sequence elements that are also present in the promoters of coordinately regulated genes. In contrast, other hypersensitive sites within the 5′-flanking sequences are expressed constitutively and one maps within an element that is also present in the promoters of coordinately regulated genes. These results suggest that the transcriptional activation of chalcone synthase genes is accompanied by structural changes in the chromatin associated with the proximal region of the promoter and that these probably reflect the binding of transcription factors tocis-regulatory elements.

Recognition and Response in Plant:Pathogen Interactions

The perception of, and response to, microbes is of crucial importance in relation to the damage and reduction in crop yield attributable to plant pathogens and the potential beneficial effects arising from symbiotic plant:microbe interactions. Our research pertaining to plant:microbe recognition is based on two premises: (a) that recognitional events result in specific responses, the study of which will lead to elucidation of signal perception and transduction mechanisms and (b) that the surfaces of microbial and plant cells are crucially involved in recognitional events. In this chapter, recent studies on the molecular mechanisms governing expression of specific plant defense responses and the molecular architecture of the surface of the phytopathogenic bacterium Pseudomonas syringae pv glycinae are discussed.

Elicitors and Defense Gene Activation in Cultured Cells

Active defense of plants against fungal or bacterial pathogens often involves the rapid death of cells in intimate contact with the invading microorganism. Accompanying this so-called hypersensitive response (HR) are rapid localized changes in host metabolism which lead to the synthesis of potential defensive barriers; these include the accumulation of low Mr antimicrobial compounds termed phytoalexins, the deposition of phenolic material and hydroxyproline-rich glycoproteins (HRGPs) in the host cell wall, and the synthesis of hydrolytic enzymes. The exact relationship between the HR and phytoalexin accumulation is still somewhat vague; in some systems, hypersensitive cell death appears to be a pre-requisite for induction and accumulation of phytoalexins in neighboring healthy cells (Bailey 1982), whereas phytoalexin synthesis can be initiated in suspension cultured cells in the absence of serious effects on cell viability (Hamdan and Dixon 1986). It is, however, well documented that molecules from plant pathogenic fungi have the ability to induce both hypersensitive-type cell necrosis and/or phytoalexin accumulation in plant cells. These so-called elicitors often originate from the fungal cell wall.

Regulation of Gene Expression in Biologically Stressed Bean Cell Cultures

For many years, an important aim of plant biotechnology has been to manipulate cultured plant cells in order to increase the metabolic flux into specific pathways of secondary product formation. In most cases this has involved alteration of physical and chemical components of the culture environment, the most common of which has been the systematic variation of the concentration of growth regulators in the culture medium. This approach has led to the design of protocols for the preliminary optimization of culture conditions for secondary product formation in specific plant species [1], although the commercial exploitation of cell cultures as sources of valuable chemicals has been disappointing [2].

Molecular Cloning of Plant Protein Kinases

Many cellular proteins are subject to post-translational modifications which alter their activity. One of the most widespread and extensively studied modifications is that of reversible phosphorylation. First demonstrated in the control of glycogen metabolism, reversible phosphorylation is now recognized as a major mechanism for regulating the activities of enzymes and proteins.